In vitro microtubule dynamics assays using dark-field microscopy

Abstract

Microtubules are dynamic non-covalent mesoscopic polymers. Their dynamic behavior is essential for cell biological processes ranging from intracellular transport to cell division and neurogenesis. Fluorescence microscopy has been the method of choice for monitoring microtubule dynamics in the last two decades. However, fluorescent microtubules are prone to photodamage that alters their dynamics, and the fluorescent label itself can affect microtubule properties. Dark-field imaging is a label-free technique that can generate high signal-to-noise, low-background images of microtubules at high acquisition rates without the photobleaching inherent to fluorescence microscopy. Here, we describe how to image in vitro microtubule dynamics using dark-field microscopy. The ability to image microtubules label-free allows the investigation of the dynamic properties of non-abundant tubulin species where fluorescent labeling is not feasible, free from the confounding effects arising from the addition of fluorescent labels.